The present invention relates to chromogenic compounds which are useful as optical indicator compounds in analytical test systems. In particular, the present invention relates to novel chromogenic enzyme substrate compounds and their use in analytical test systems for the detection of enzymes in a liquid test sample.
The determination of enzymes is important in a variety of fields such as biochemical research, environmental and industrial testing, and medical diagnostics. The quantitation of enzyme levels in body fluids such as serum and plasma provides very useful information to the physician in diagnosing diseased states and their treatment. In addition to being analytes of interest in biological fluids, enzymes can also serve as detection reagents in a variety of analytical systems such as immunoassays and nucleic acid hybridization techniques. In such systems, enzymes are useful directly or indirectly as labels to monitor the extent of antigen-antibody binding or nucleic acid hybridization that occurs.
Accordingly, the desire to detect enzyme analytes and to use enzyme labels as a diagnostic tool in various analytical test systems has given rise to the development of optical indicator compounds for use in the detection and measurement of the activity of such enzymes. Typically, such known optical indicator compounds comprise a detectable chemical group, such as a fluorogen or a chromogen, which has been derivatized with an enzyme cleavable substrate group specific for the enzyme of interest. Such optical indicator compounds exhibit an optical signal which is different from the optical signal which is provided by the cleaved native form of the fluorogen or chromogen. In principle, the enzyme cleaves the indicator compound to liberate the fluorogen or chromogen in the form of a distinctly fluorescent or colored product to provide a change in fluorescence or color which is proportional to the amount of enzyme present which, in turn, can be correlated to the amount of analyte present in a liquid test sample.
In particular, the detection and/or determination of hydrolases, i.e., enzymes which catalyse hydrolysis reactions of esters, glycosidic bonds, peptide bonds, other carbon-nitrogen bonds, and acid anhydrides [see Lehninger, Biochemistry (Worth Publishers, Inc., New York, N.Y., 1970) p. 148], is of interest in the diagnosis and monitoring of various diseases such as, for example, the determination of amylase and lipase in the diagnosis of pancreatic dysfunction [see Kaplan and Pesce, Clinical Chemistry--Theory, Analysis and Correlation (C. V. Mosby Co., St. Louis, Mo., 1984) Chapter 56], determination of N-acetylglucosaminidase (NAG) as an indicator of renal disease [see Price, Curr. Probl. Clin. Biochem.9, 150 (1979)], and detection of esterase as an indicator for leukocytes [see Skjold, Clin. Chem. 31, 993 (1985)]. Further to their value in disease monitoring, hydrolases in recent years have gained importance in the diagnostic as well as in the biotechnology areas. For example alkaline phosphatase and, preferably, .beta.-D-galactosidase have found increasing use as indicator enzymes for enzyme immunoassays [see Annals of Clinical Biochemistry 16, 221-40 (1979)].
Accordingly, the use of enzymes such as glycosidases, particularly .beta.-D-galactosidase, as indicator enzyme labels in analytical test systems has given rise to the development of substrate glycosides such as phenyl-.beta.-D-galactoside, o-nitrophenyl-.beta.-D-galactoside and p-nitrophenyl-.beta.-D-galactoside [see Biochem. Z., Vol. 333, p. 209 [1960)] which are hydrolysed by .beta.-D-galactosidase to liberate the phenols which are determined photometrically in the ultraviolet range, or the nitrophenols which are determined in the shortwave visible range, respectively. European Patent Publication No. 156,347 and U.S. Pat. No. 4,810,636 describe glycosides of resorufin and acridinone derivatives, respectively, which are specific for and cleaved by the particular glycosidase of interest to liberate detectable chromogens. U.S. Pat. No. 3,950,322 describes an N-acyl-neuraminic acid derivatized with a fluorogen such as 4-methylumbelliferone, fluorescein, methylfluorescein, resorufin, or umbelliferone for the detection of neuraminidase where the fluorogenic substrate glycoside is similarly acted upon by the enzyme to liberate the fluorogen.
The use of .beta.-D-galactosides has also been described in conjunction with histochemical investigations, such as the napthyl-.beta.-D-galactosides described in Histochemie, Vol. 35, p. 199 and Vol. 37, p. 89 (1973), and the 6-bromo-.alpha.-napthyl derivatives thereof described in J. Biol. Chem., Vol. 195, p. 239 (1952). According to such test systems, the napthols which are liberated upon the interaction of the galactoside with the enzyme are reacted with various diazonium salts to yield the respective azo-dyes which can then be visualized.
Although such known optical indicator compounds are useful for the detection of enzyme analytes and labels in an analytical test system, a number of problems nevertheless exist which effect assay sensitivity and accuracy such as low extinction coefficients, poor water solubility, absorbance maxima which interfere with various pigments and other constituents commonly present in biological fluids, and color shifts between the optical indicator compound and the liberated chromogen or fluorogen which are difficult to measure without the use of complicated instruments.
Accordingly, it is an object of the present invention to provide chromogenic enzyme substrate compounds which can be employed as optical indicator compounds in analytical test systems for the accurate and sensitive determination of enzymes in a liquid test sample.
Further, it is an object of the present invention to provide chromogenic enzyme substrate compounds which can be incorporated into the solid, porous matrix of an analytical test device as optical indicator compounds for the measurement of enzymes incorporated therein or in a liquid test sample applied thereto.